1990 Fiscal Year Final Research Report Summary
Transfection of Multidrug Resistant Gene into Human Hematopoietic Stem Cells and its Clinical Application in Bone Marrow Transplantation
| Project/Area Number |
01480299
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | Nagoya University |
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Principal Investigator |
OHNO Ryuzo Nagoya University Associate Professor Department of Medicine, 医学部, 助教授 (70093002)
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| Co-Investigator(Kenkyū-buntansha) |
NAOE Tomoki Nagoya University Assistant Professor Department of Medicine, 医学部, 助手 (50217634)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Gene transfection / Multidrug resistant gene / MDR1 / P-glycoprotin / Hematopoietic stem cells / Retroviral vector / Drug resistance / Phenotypic change |
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| Research Abstract |
Multidrug Resistant gene (MDR1) encodes a membrane-binding P-glycoprotein (approximately 170 kDa molecular mass) that plays an important role in efflux transportation of structurally unrelated cytotoxic drugs such as Vinca alkaloids, anthracycline, colchicine, actinomycin D and some other drugs, some of which are now clinically used in cancer chemotherapy.
Recently, isolation of the gene for MDR1 has raised hope that multidrug resistance of cancer cells might be elucidated at a molecular level.
We aimed to introduce MDR1 gene into human hematopoietic stem cells to obtain otherwise normal blood cells with a multidrug resistant phenotype.
MDR1 cDNA fragment (4.3 kb in length) was cloned from cDNA library derived from monoblastic leukemia cell line, NOMO1/ADR. This cDNA has been inserted into a retroviral vector pCO1. Virus was produced after transfection of this vector into a packaging cell line PA-12. This virus conferred multidrug resistant phenotype on dog kidney cell line MDCK.
Twenty to 30-hold concentration of human multipotent stem cells from normal bone marrow was achieved by panning and sorting protocol with monoclonal antibodies. Virus containing MDR1 cDNA was infected into these cells. There is no increase in the number of colonies cultured with drug-containing medium. Increment of viable cells in surviving colonies was observed.
We preliminarily analyzed a potential use of new expression system. Fifty-hold amplification of virus titer was accomplished in the producer cells. However, a large deletion of viral genome was also identified, suggesting that the gene defect might be caused by a rapid gene duplication.
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